Hairless

DEVELOPMENTAL BIOLOGY

Embryonic

Initially, Hairless transcripts are detected in a uniform distribution in embryos until the blastoderm stage; this pattern most likely corresponds to maternal mRNA deposition in the egg. With the onset of gastrulation, zygotic expression appears in the entire germ band, where mRNA clearly accumulates basally in the cells. During germ band extension Hairless transcripts rapidly fade from the ectodermal cell layer and become enriched in the mesodermal sheath and in the invaginating anterior and posterior midgut. With the flattening of the mesoderm, this staining becomes less evident, but can be still detected. Midgut staining is most prominent particularly in older embryos where expression also in the central nervous system and body wall is discernible, but it is uncertain whether this body wall staining represents cells of ectodermal or mesodermal origin (Maier, 1992).

Maternal H transcripts accumulate in ovarian nurse cells. These transcripts are present in syncytial embryos and persist until the cellular blastoderm stage. The transcript that begins to accumulate during late gastrulation and early germ-band extension evidently represents the onset of zygotic H expression. This interpretation is consistent with the appearance at 4-6 hr of a novel 6.0 kb transcript that is not present earlier. These zygotic transcripts are broadly distributed in the embryo throughout germ-band extension and retraction, although initially they appear to accumulate at a somewhat higher level in the mesodermal layer, whereas lower levels are observed in parts of the head region, especially the procephalic lobe and the clypeolabrum. H transcripts are present in the developing CNS at the time the zygotic neurogenic genes become active, consistent with the suppression by H mutations of the neural hyperplasia caused by loss of neurogenic gene function (Bang, 1992 and references).

Larval

There is a widespread, apparently uniform distribution of H transcripts at the time of macrochaete SOP determination. Within the first 16 hours after puparium formation, H transcripts appear to be uniformly distributed in the notum epithelium, except for a persistently higher level of accumulation in two cells of the developing macrochaetes. By 16 hr after puparium formation, in a background of generalized expression, higher levels of transcript are detectable in single cells and, possibly, pairs of cells in the positions of the future microchaetes. These cells may represent the secondary precursors that will generate the trichogen and tormogen: such precursors are present and known to be about to commence their division at this time (Bang, 1992).

Effects of Mutation or Deletion

A number of genetic loci, called neurogenic, have been found to be involved in directing the segregation of neural and epidermal lineages within the ectodermal germ layer of Drosophila. With the aim of understanding the regulation of this developmental function, interactions among the loci N, Dl and E(spl) were studied, as were interactions with another locus (Hairless), by means of increasing and decreasing the number of wild-type copies of any one of these genes in the presence of mutations in any other one. The results reveal a functional community that exists among these neurogenic loci. E(spl) overlaps functionally with both N and Dl because genotypes involving only one copy of E(spl)+ and either an N or Dl mutation are lethal. Furthermore the normal H+ allele behaves as if it represses the activity of the 3 neurogenic loci, and whereas E(spl) seems to be a close target of H repressive action, the influence of H on the other two seems to be indirect (Vassin, 1985).

Reduction of the wild-type activity of the gene Hairless (H) results in two major phenotypic effects on the mechanosensory bristles of adult Drosophila. Bristles are either 'lost' (i.e. the shaft and socket fail to appear) or they exhibit a 'double socket' phenotype, in which the shaft is apparently transformed into a second socket. Analysis of the phenotypes conferred by a series of H mutant genotypes demonstrates (1) that different sensilla exhibit different patterns of response to decreasing levels of H+ function, and (2) that the 'bristle loss' phenotype results from greater loss of H+ function than the 'double socket' phenotype. The systematic study of H allelic combinations enabled the identification of genotypes that reliably produce specific mutant defects in particular positions on the bodies of adult flies. This permitted an investigation of the cellular development of sensilla in these same positions in larvae and pupae and thereby established the developmental basis for the mutant phenotypes. H is required for at least two steps of adult sensillum development. In positions where 'double socket' microchaetes appear on the notum of H mutant flies, sensillum precursor cells are present in the developing pupa and divide normally, but their progeny adopt an aberrant spatial arrangement and fail to differentiate correctly. In regions of the notum exhibiting 'bristle loss' in adult H mutants, at the appropriate stages of development to detect sensillum-specific cell types, neither the precursor cell divisions that generate them, nor the primary precursor cells themselves could be detected. Thus, the H 'bristle loss' phenotype appears to reflect a very early defect in sensillum development, namely the failure to specify and/or execute the sensory organ precursor cell fate. This finding indicates that H is one of a small number of identified genes for which the loss-of-function phenotype is the failure of sensillum precursor cell development (Bang, 1991).

Overexpression of H produces two bristle shafts in the position of mechanosensory bristles, with no socket. This defects is interpreted as a tormogen-to-tricogen cell transformation, which is the opposite transformation from that which underlies the H hypomorphic double socket phenotype. In addition, adult flies developed from heat-shocked H overexpression exhibit a high frequency of multiplication and/or loss of microchaetes or macrochaetes. These phenotypes strongly mimic those caused by loss-of-function mutations of the neurogenic genes (Bang, 1992).

The mechanosensory bristles of adult Drosophila are composed of four cells that, in most cases, are the progeny of a single sensory organ precursor (SOP) cell. Two sister cells in this lineage, the trichogen and tormogen, produce the external shaft and socket of the bristle, respectively. Loss-of-function mutations of Hairless (H) confer two distinct mutant phenotypes on adult bristles. The bristle loss phenotype results from the failure to specify and/or execute the SOP cell fate; the double socket phenotype results from the transformation of the trichogen (shaft) cell into a second tormogen (socket) cell. The H gene encodes a novel basic protein with a predicted molecular mass of 109 kD. Basal levels of expression of a transgene (P[Hs-H]) in which the H protein-coding region is under the control of the Hsp70 promoter are sufficient to provide full rescue of H mutant phenotypes. Heat shock treatment of P[Hs-H] transgenic animals as late larvae and early pupae produces a tormogen-to-trichogen (double shaft) cell fate transformation, as well as bristle multiplication and loss phenotypes very similar to those caused by loss-of-function mutations in the neurogenic gene Notch. These results indicate that the SOP cell fate requires H to antagonize the activity of the neurogenic group of genes and that the expression of distinct cell fates by the trichogen/tormogen sister cell pair depends on an asymmetry in their levels of H+ activity or in their thresholds for response to H (Bang, 1992).

Successive alternative cell fate choices in the imaginal disc epithelium lead to the differentiation of a relatively invariant pattern of multicellular adult sensory organs in Drosophila. The activity of Suppressor of Hairless is required for both the sensory organ precursor (SOP) versus epidermal cell fate decision, and for the trichogen (shaft) versus tormogen (socket) cell fate choice. Complete loss of Suppressor of Hairless function causes most proneural cluster cells to accumulate high levels of the Achaete and Delta proteins and to adopt the SOP fate. Late or partial reduction in Suppressor of Hairless activity leads to the apparent transformation of the tormogen (socket) cell into a second trichogen (shaft) cell, producing a 'double shaft' phenotype. Overexpression of Suppressor of Hairless has the opposite phenotypic effects. SOP determination is prevented by an early excess of Suppressor of Hairless activity, while at a later stage, the trichogen (shaft) cell is transformed into a second tormogen (socket) cell, resulting in 'double socket' bristles. It is concluded that, for two different cell fate decisions in adult sensory organ development, decreasing or increasing the level of Suppressor of Hairless function confers mutant phenotypes that closely resemble those associated with gain and loss of Hairless activity, respectively. These results, along with the intermediate SOP phenotype observed in Suppressor of Hairless;Hairless double mutant imaginal discs, suggest that the two genes act antagonistically to stably commit imaginal disc cells to alternative fates (Schweisguth, 1994).

In Drosophila imaginal discs, the function of the Hairless (H) gene is required at multiple steps during the development of adult sensory organs. Reported is a series of experiments designed to investigate the in vivo role of H in sensory organ precursor (SOP) cell specification. The proneural cluster pattern of proneural gene expression and of transcriptional activation by proneural proteins is established normally in the absence of H activity. By contrast, single cells with the high levels of achaete, scabrous, and neuralized expression characteristic of SOPs almost always fail to appear in H mutant proneural clusters. These results indicate that H is required for a relatively late step in the development of the proneural cluster, namely, the stable commitment of a single cell to the SOP cell fate. Expression of an activated form of the Notch receptor leads to bristle loss with the same cellular basis (failure of SOP determination) as loss of H function. Simultaneous overexpression of H suppresses this effect. Epistasis experiments demonstrated that the failure of stable commitment to the SOP fate in H null mutants requires the activity of the genes of the Enhancer of split complex, including groucho. These results indicate that H promotes SOP determination by antagonizing the activity of the Notch pathway in this cell, thereby protecting it from inhibitory signaling by its neighbors in the proneural cluster. A simple threshold model is proposed in which the principal role of H in SOP specification is to translate a quantitative difference in the activity of the Notch pathway (in the SOP versus the non-SOP cells) into a stable binary cell fate decision (Bang, 1995).

A single copy of Hairless, is able to suppress the wing defects of heterozygous strawberry notch, suggesting that Hairless and sno exhibit related antagonistic activities downstream of the Notch pathway. In a similar fashion, a single copy of Suppressor of Hairless and a single copy of sno show enhanced defects, indicating that Su(H) and Sno cooperate closely in patterning the wing. As Su(H) and Sno have not been shown to physically interact, this may mean that the two proteins work in parallel or that the interaction is too weak to be detected (Majumdar, 1997).

Hairless full length cDNA constructs give a robust rescue of H haplo-insufficiency phenotypes. In order to determine the respective wild type Hairless activity, deletion constructs were tested for their ability to restore loss of macrochaetae on head and notum and the wild type wing venation. Hairless derivatives are heavily impaired in their rescue capacity. Even a small deletion (the C4 deletion in the C-terminal protein of the protein) causes a dramatic drop in performance. Reduction of H activity is also observed in either N- or C-terminally truncated constructs, C1 and C6. C1, lacking the N-terminal amino acids has nearly lost all H wild type activity, while C6, lacking the 3' untranslated region and the 15 C-terminal amino acids, gives measurable rescue. Unexpectedly, the C3 construct in which the central acidic domain is deleted restores the number of bristles even better than full length H. This central domain, termed the A-domain, may mark a repression domain of the Hairless protein required for silencing Hairless function, e.g. for releasing Su(H) from a H/Su(H) complex. Loss of the N-terminal domain results in a construct that no longer rescues Hairless loss of function mutants yet is still able to induce gain of function phenotypes and, in addition, shows normal Su(H) binding in an in vitro assay. Apparently, the N-domain harbors sequences crucial for H wild type function, separable from the adjacent S-domain, required for binding of Su(H). It is conceivable that the N-domain is involved in the inhibition of Su(H) DNA binding and/or trans-activation, whilst the S-domain might represent mainly the H-Su(H) interactive surface (Maier, 1997).

Drosophila Hairless (H) encodes a negative regulator of Notch signaling. H activity antagonizes Notch (N) signaling during bristle development at the pupal stage. Clonal analysis reveals that H acts by inhibiting signal transduction rather than by promoting signal production, during both selection of microchaete precursors in the notum and vein cell differentiation in the wing. Allele-specific interactions further suggest that H inhibits Notch signal transduction by interacting directly with Suppressor of Hairless. Unexpectedly, this regulatory function of H appears to be essential only during imaginal development. Using a null allele of H that corresponds to a deletion of the H coding sequence, it has been shown that embryos devoid of both maternal and zygotic gene products develop similar to wild-type embryos. Thus, H activity is not strictly required to regulate N-mediated cell fate choices in the embryo (Schweisguth, 1998).

Quantitative trait loci (QTL) affecting response to short-term selection for abdominal bristle number have been mapped to seven suggestive regions that contain loci involved in bristle development and/or that have adult bristle number mutant phenotypes, and are thus candidates for bristle number QTL in natural populations. To test the hypothesis that the factors contributing to selection response genetically interact with these candidate loci, high and low chromosomes from selection lines were crossed to chromosomes containing wild-type or mutant alleles at the candidate loci, and the numbers of bristles were recorded in trans heterozygotes. Quantitative failure to complement, detected as a significant selection line*cross effect by analysis of variance, can be interpreted as evidence for allelism or epistasis between the factors on selected chromosomes and the candidate loci. Mutations at some candidate loci (bb, emc, h, Dl, Hairless) show strong interactions with selected chromosomes, whereas others interact weakly (ASC, abd, Scr) or not at all (N, mab, E(spl)). These results support the hypothesis that some candidate loci, initially identified through mutations of large effect on bristle number, either harbor or are close members in the same genetic pathway as variants that contribute to standing variation in bristle number (Long, 1996).

Formation of mechano-sensory organs in Drosophila involves the selection of neural precursor cells (SOPs) mediated by the classical Notch pathway in the process of lateral inhibition. The subsequent cell type specifications rely on distinct subsets of Notch signaling components. Whereas E(spl) bHLH genes implement SOP selection, they are not required for later decisions. Most remarkably, the Notch signal transducer Su(H) is essential to determine outer but not inner cell fates. In contrast, the Notch antagonist Hairless, thought to act upon Su(H), strongly influences the entire cell lineage, demonstrating that it functions through targets other than Su(H) within the inner lineage. Therefore, Hairless and Numb may have partly redundant activities. This suggests that Notch-dependent binary cell fate specifications involve different sets of mediators depending on the cell type considered (Nagel, 2000a).

Hairless is an important antagonist of Notch signaling throughout the mechano-sensory organ lineage As suggested by the enrichment of H protein within all four cells of an emerging bristle, it is a potent antagonist of Notch signaling within this entire cell lineage. H protects the sensory organ precursor from a Notch signal. In the absence of H nearly all of the SOPs are forced into a non-neuronal fate. The consequence is a pronounced bristle loss. H is absolutely essential within shaft cells and the neuron and is important for the pIIb (precursor of a glial cell and of the pIIIa, the precursor of the sheath and neuron cells) as well, to allow for proper cell fate choices. Changing the dose of H affects all three levels of sensory organ development. However, achievement of the first level, emergence of the sensory organ precursor, is a prerequisite for the development of its progeny. Thus, the number of descendants that can be assessed for requirement of H activity is naturally small in a strong H compromised background. In accounting for this obstacle, a phenotypic series of H mutants has been analyzed and different sensitivities of external and internal cell fate decisions have been found: whereas the socket/shaft transformation is always complete, that of neuron to sheath appears gradual when looking in the hypomorphic condition. Only in a strong loss of function condition like in H amorphs, is the transformation complete. In the gain of function situation, the transformation is always complete in a wild-type background, where extra neurons arise at the expense of sheath cells, whereas a gradual transition is seen when deltex activity is elevated simultaneously. This finding suggests that the two internal cells, sheath cell and neuron, are not equivalent, in that the neuronal fate appears to be more stable over the thecogen fate dependent on deltex activity. Another example of such a bias is found in the decision between internal and external cell fate, where pIIb is clearly preferred over pIIa. In both cases, Notch signaling might be rather weak between the two cells, thus favoring the ground state (Nagel, 2000a).

The decision between the tormogen (socket) versus trichogen (shaft) fate of the pIIa progeny seems to depend strictly on the balanced doses of H and Su(H). Changes in the dose of either one pushes the equilibrium completely towards the opposite fate. Accordingly, Su(H) protein accumulates to very high levels in the future tormogen, and can thus override the elevated levels of H protein within this cell. The epistasis of H over dx regarding outer bristle cell fates can be easily explained by the dominating activity of Su(H) within the pIIa progeny. The choice between neuron and thecogen (sheath) fate is based on a quite different mechanism, because unlike H, Su(H) is not necessary for the emergence of the two opposing cell types. The default state of pIIIb descendants is neuronal. The Notch signal redirects one of these cells into thecogen fate. Although both Su(H) and dx, positively influence Notch signaling in the presumptive thecogen, none of the two is required for the generation of this cell type. Thus, the Notch signal in the thecogen might be transduced by a molecular mechanism independent of Su(H) or dx involving as yet unknown factor(s). In the absence of H, the presumptive neuron gains thecogen fate. Therefore, H has an important role in protecting the neuron from the Notch signal. Since this signal does not emanate from Su(H), H must act through unknown component(s). This is the first unambiguous example of a Su(H)-independent function of H (Nagel, 2000a).

Earlier work has identified numb as an important factor during binary cell fate decisions within the sensory organ lineage, where it is partitioned into those cells in which it acts as intrinsic inhibitor of the Notch signal. Both loss and gain of numb activity causes cell transformations very similar to H except for the selection of the SOP during lateral inhibition, which is controlled by H and not by numb. It is noted, however, that phenotypes are always much more penetrant when H is involved in comparison to numb. H mutations act as dominant enhancers of numb mutations, and the two proteins might physically interact with each other. Both activities are required for normal bristle development because loss of function of either gene gives a similar phenotype regarding cell type specification. To what degree do these activities overlap? Close inspection of the phenotypes shows that numb has a very strong influence on the pIIa/pIIb fate selection but less on the subsequent binary decisions because cell type transformations are always partial. Although this might be a quantitative difference, it is noted that H activity is strictly required to guard both shaft cell and neuron from a faulty Notch signal and that complete cell type transformations are observed as a consequence of H activity loss. Furthermore, H activity is also required during the process of lateral inhibition, suggesting that H is the more general antagonist in Notch-dependent processes (Nagel, 2000a).

Both in loss of function and gain of function combinations numb is epistatic to Su(H) within the pII cells, indicating that Su(H) acts downstream of numb. This is in agreement with a model, whereby numb antagonizes Notch signaling through direct interference with the Notch receptor. Within the pIIa progeny, however, Su(H) can override the inhibiting activity of ectopic numb protein. This interference might again be direct, because preliminary evidence from yeast interaction trap experiments shows that Numb and Su(H) physically interact. Furthermore, by inhibiting Notch signaling, numb might indirectly modulate the levels of Su(H) transcriptional activity. Thus, the conflicting epistasis data might reveal once more different sensitivities of the sensory organ cell lineage regarding Notch signaling, especially the preference of the pIIb over the pIIa fate (Nagel, 2000a).

During the development of mechanosensory organs, Notch is required at two distinct steps: the singling out of the sensory organ precursor, SOP, and the correct specification of cell fates within the sensory organ lineage, SOL. Apparently, different subsets of Notch signaling components are used for these two processes. Whereas SOP selection in the process of lateral inhibition requires the 'classical' battery of Notch signaling components, namely Su(H), dx, mam, E(spl) bHLH and H, the subsequent asymmetric cell divisions require only certain Notch components plus the intrinsic activity of numb. Numb plays a major role in the distinction between the pII siblings. In the pIIa progeny, socket cell fate is enforced with the help of Su(H) and, to a lesser degree, dx, and the role of H is to protect the shaft from this fate. In the sub-epidermal progeny, Notch signaling determines sheath cell fate, promoted to some degree by dx and Su(H). However, since neither of the components, Su(H), dx nor E(spl) bHLH are strictly required for the selection of thecogen fate, the Notch signal is transduced by other factor(s), X. The role of mam in this process is as yet undecided, since the mutant cell clones are rather uninformative and appropriate overexpression constructs are unavailable. The neuron has to be protected from the Notch signal, and both numb and H play a pivotal role in this process. Apparently, the target of numb is the Notch receptor itself. It is not clear whether H acts at the same level, or whether it acts on a different target, maybe directly involving the presumptive signal transducer X. Although both mam and dx might be targets of H, no physical interactions were observed in the yeast interaction trap assay. Overall, H represents a key player in antagonizing the Notch signal and thus assures, that in the end all four different cell types of the mechano-sensory organ arise (Nagel, 2000a).

A summary of Notch signaling during mechano-sensory organ development is presented. Notch signaling is required in the entire cell lineage, as is the antagonist Hairless. Whereas the lateral inhibition process uses the classical battery of Notch signaling components, the subsequent binary cell fate specifications rely only on a subset of these components and involve in addition the intrinsic antagonist Numb. In a first step SOP is singled out by lateral inhibition from a proneural cluster, protected through the activity of H. The surrounding cells are forced by the SOP into epidermal fate through the activation of the Notch receptor, implemented with the help of Su(H), dx, mam and E(spl) bHLH proteins (classical pathway). In a second step, Notch signal, promoted by Su(H) and dx, forces one SOP daughter cell into pIIa fate from which the pIIb cell is protected by the antagonists numb and H. The pIIb gives rise to the pIIIb and a glial cell. In a third step, the progeny of pIIa are socket and shaft cell. The socket cell receives the Notch signal via Su(H) and dx, the effector genes are unknown. The shaft cell is protected by H and numb from the Notch signal. In a fourth step, the progeny of the pIIIb are sheath cell and neuron. The sheath cell receives a Notch signal promoted by unknown factor X, whereas the neuron is protected by H and numb (Nagel, 2000a).

Overexpression of m4/alpha (see E(spl) region transcript m4) blocks lateral inhibition. The SOP pattern is a result of an interplay between proneural proteins, which promote neural fate, and Notch signaling, which inhibits it. The fact that supernumerary chaetael/SOPs arise in close apposition to each other suggests that the contact-dependent Notch signaling that normally counters SOP fate may be compromised. To locally block lateral inhibition for comparison purposes, a well characterized negative regulator of Notch signaling, Hairless (H) was overexpressed. H is known to negatively modulate Notch signaling by interfering with the activity of the transcriptional activator Su(H), by which at least part of the Notch signal is transduced to the nucleus (Apidianakis, 1999).

Generally, the effect of H overexpression is similar to that of m4/alpha. At the ACV (anterior cross-vein campaniform sensillum), L3 (third longitudinal vein campaniform sensillum) and wing margin clusters, the extent of SOP overcommitment is comparable to that caused by m4/alpha, whereas at the dorsal radius H gives much higher numbers of supernumerary SOPs. The effects of H differ from those of m4/alpha in two further respects. (1) H abolishes some wing margin sensilla, presumably by interfering with the Su(H)-dependent inductive Notch signaling that sets up the dorsoventral boundary, which subsequently induces margin SOPs. m4/alpha does not affect the process of dorsoventral wing patterning, consistent with the presence of a full complement of margin SOPs. (2) In the adult phenotype, whereas m4/alpha produces solely bristle tufting, H overexpression variably produces naked patches or double-shaft socketless bristles, consistent with its proposed role in the SOP lineage cell fate decisions. Ectopic expression of m4/alpha gives neither of these phenotypes, suggesting that it affects only SOP singularization but not the SOP lineage. In order to test this hypothesis, pupal nota were stained with antibodies directed against Elav, a neuron specific marker, and Pros, specific to the sheath cell. There is a one-to-one correspondence between Elav positive and Pros positive cells. Therefore, overexpression of m4/alpha does not upset the Notch/Numb mediated asymmetric divisions in the SOP lineage. The only step in sensory organ development that m4/alpha seem to affect is that of lateral inhibition, which restricts the number of SOPs produced per proneural cluster (Apidianakis, 1999).

Experiments by Nagel (2000), suggest that the overexpression phenotype of E(spl) m4 and E(spl) malpha obtained by Apidianakis (1999) is likely to be due to a dominant negative effect and does not reflect the biological function of these two genes. In order to elucidate m4/malpha gene function directly, RNAi, which causes sequence-specific transcript degradation, was carried out by injecting either m4 or malpha double-stranded RNA or a mixture of both into pre-blastoderm embryos. In agreement with genetic data, RNAi causes a high incidence of lethality (~50%). Dead embryos develop intermediate to strong neurogenic phenotypes (too many neurons) typical of loss of E(spl) bHLH activity. Surviving embryos hatch into wild type appearing larvae that develop normally to adult flies. From this it is concluded that the m4/malpha genes are required to positively transduce the Notch signal during neurogenesis, and presumably during bristle development as well. Therefore, suppression of lateral inhibition observed after overexpression of either m4/malpha family member must be due to a dominant-negative effect, presumably by titrating out other important Notch pathway components (Nagel, 2000b).

ro^Dom is a dominant allele of rough (ro) that results in reduced eye size due to premature arrest in morphogenetic furrow (MF) progression. The ro^Dom stop-furrow phenotype is sensitive to the dosage of genes known to affect retinal differentiation, in particular members of the hedgehog (hh) signaling cascade. ro^Dom interferes with Hh's ability to induce the retina-specific proneural gene atonal (ato) in the MF and normal eye size can be restored by providing excess Ato protein. ro^Dom was used as a sensitive genetic background in which to identify mutations that affect hh signal transduction or regulation of ato expression. In addition to mutations in several unknown loci, multiple alleles of groucho (gro) and Hairless (H) were recovered. Analysis of their phenotypes in somatic clones suggests that both normally act to restrict neuronal cell fate in the retina, although they control different aspects of ato's complex expression pattern (Chanut, 2000).

Loss-of-function ro mutations cause eye roughness, due to mis-specification of photoreceptors R2 and R5, and the formation of ommatidia with more than one R8 photoreceptor. Repression of R8 cell fate has been attributed to inhibition of ato expression by the Ro homeodomain protein. In support of this proposal, Rough and Atonal proteins appear in complementary sets of cells behind the MF, and ato expression is expanded behind the MF in ro mutants. Generalized expression of ro under a heat-shock promoter (hs-ro) leads to loss of ato expression in the MF and eventually results in furrow arrest. Furrow arrest in ro^Dom is also accompanied by loss of ato expression in the MF. By analogy to the hs-ro phenotype, it is proposed that ro^Dom leads to excess Ro production, although that excess is not detectable by antibody staining (Chanut, 2000).

ro^Dom is very sensitive to alterations of ato gene dosage, since it is enhanced by loss-of-function ato alleles and almost completely rescued when high levels of ato expression are restored ahead of the MF. The ro^Dom phenotype therefore appears to result primarily from inhibition of ato expression due to excess Ro protein. On the basis of this understanding, the role of two of the strongest suppressors isolated in this screen, new alleles of gro and H, were analyzed on ato regulation and furrow progression (Chanut, 2000).

Hairless inhibits N signaling by preventing Su(H), a transcription factor, from translocating to the nucleus and activating transcription of N targets such as the E(spl) complex genes. In the absence of H, Su(H) is free to enter the nucleus upon activation of N. Su(H) mutant clones lead to expanded Ato expression behind the MF, consistent with a role for Su(H) in the N-mediated lateral inhibition that leads to the refinement of ato expression. In H clones are found in which the refinement of ato expression to single cells appears accelerated. This is consistent with a role for H as an inhibitor of N and Su(H) in lateral inhibition. Surprisingly, however, individual clusters of Ato-expressing cells often persist in H mutant tissue behind the MF, instead of resolving to single R8 precursors; in adults as well, mutant ommatidia often contain more than one R8. This would suggest that at later stages H is required to resolve ato expression to single R8 precursors, a role that is not expected for an inhibitor of the N pathway. Anterior H mutant clones show precocious ato expression anterior to the MF. This might explain the patterning defects behind the MF, if precocious and excessive accumulation of Ato protein in the MF interferes with the proper execution of lateral inhibition via N or with downregulation by Ro. In this regard, it is noted that excess Ato protein, as provided under heat-shock control, is found to perturb the resolution of ato expression to single R8 precursors (Chanut, 2000).

It has been suggested that early ato expression, ahead of the MF, is in part the result of an as yet unsuspected 'proneural' effect of N signaling. The anterior expansion of ato expression in H mutant clones is consistent with this model, assuming that H would act as an inhibitor of N there as well. However, the proneural function of N must not be mediated by Su(H), since removal of Su(H) function does not abolish ato expression ahead of the MF. The results presented here may indicate that H antagonizes the proneural function of N via a mechanism that does not involve Su(H). Alternatively, the role of H on early ato expression may be independent of N signaling. Regardless of the exact mechanism, the enhanced expression of ato ahead of the MF in H mutants is likely to explain suppression of the ro^Dom phenotype by counteracting the effect of ectopic Ro on ato expression in the MF (Chanut, 2000).

Finding that similar levels of suppression can be achieved by loss-of-function mutations in H and gro (which act in opposite directions in the N pathway) is not unique. A similar situation was encountered in another study where mutations in gro and H were both found to enhance the wing and bristle phenotypes associated with loss-of-function mutations in Egfr. The observation that mutations in both genes elevate ato expression in the vicinity of the MF, but at different stages of the differentiation process, helps resolve this paradox. The results also indicate that the exact timing (or location) of ato expression might not be crucial to MF progression, provided adequate levels are reached. This conclusion is supported by the finding that Ato supplied anterior to its normal expression domain, in the h expression domain, restores normal eye size in a ro^Dom background. Whether proper R8 spacing and ommatidial patterning can be achieved under these conditions remains to be shown (Chanut, 2000).

The Notch pathway is known to act during initiation and differentiation of wing veins to refine the adult vein pattern. Since nemo mutant, nmo^adk, was identified as a modifier of Notch in the eye, the link between nmo and Notch signaling in the wing was investigated. Genetic interactions between nmo^adk and mutations in several components of the Notch pathway were characterized. Mutations in the ligand Delta (Dl/+) cause a mild vein thickening phenotype. This mutation is synergistically enhanced by homozygosity for nmo^adk. Conversely, mutations in the negative regulator Hairless (H/+), which normally exhibit shortening of LV, suppress the ectopic veins seen in nmo^adk. In addition to interactions in wing veins, H and nmo^adk show a synergistic interaction in the macrochaete bristles of the head and notum (Verheyen, 2001).

nmo^adk flies have a mild bristle loss phenotype, and occasionally display bent bristles or duplicated bristles. H/+ flies display a characteristic dominant loss of macrochaetes. Homozgosity for nmo^adk in a H/+ background leads to a dramatic enhancement of the H/+ bristle loss phenotype. Since nmo^adk mutations are enhanced by Dl, and are suppressed in the wing by H, whether nmo acts upstream of Notch was examined. It was asked if the nmo^adk extra vein defect could be rescued through ectopic activation of Notch signaling. Delta and E(spl)mß were ectopically expressed with the 32B-Gal4 driver, which is expressed in the wing blade. E(spl)mß is normally expressed in the cells flanking the presumptive veins and acts to suppress rhomboid expression to the narrow band of vein progenitors. Ectopic expression of UAS-E(spl)mß leads to mild vein thinning and a shortening of LV. Both UAS-Delta and UAS-E(spl)mß specifically suppress the extra veins associated with nmo^adk mutations. Thus, both ectopic activation of the Notch pathway and loss of a negative regulator as seen with H¹/+ can lead to suppression of ectopic veins caused by nmo^adk. These results suggest that Nemo is upstream of Notch and acts in a common vein regulatory pathway (Verheyen, 2001).

In the developing Drosophila eye, cell fate determination and pattern formation are directed by cell-cell interactions mediated by signal transduction cascades. Mutations at the rugose locus (rg) result in a rough eye phenotype due to a disorganized retina and aberrant cone cell differentiation, which leads to reduction or complete loss of cone cells. The cone cell phenotype is sensitive to the level of rugose gene function. Molecular analyses show that rugose encodes a Drosophila A kinase anchor protein (DAKAP 550). Genetic interaction studies show that rugose interacts with the components of the Egfr- and Notch-mediated signaling pathways. These results suggest that rg is required for correct retinal pattern formation and may function in cell fate determination through its interactions with the Egfr and Notch signaling pathways. rugose has also been identified in a genetic screen for modifiers of Hairless (H), a Notch pathway antagonist (Schreiber, 2002) and rugose interacts with Egfr and N signaling pathways (Shamloula, 2002).

Hairless was identified as antagonist in the Notch signaling pathway based on genetic interactions. Molecularly, Hairless inhibits Notch target gene activation by directly binding to the Notch signal transducer Su(H). Additional functional domains apart from the Su(H) binding domain, however, suggest additional roles for the Hairless protein. To further understanding of Hairless functions, a genetic screen was performed for modifiers of a rough eye phenotype caused by overexpression of Hairless during eye development. A number of enhancers were identified that comprise mutations in components of Notch- and Egfr-signaling pathways, some unknown genes and the gene rugose. Mutant alleles of rugose display manifold genetic interactions with mutants in Notch and Egfr signaling pathway components. Accordingly, the rugose eye phenotype is rescued by Hairless and enhanced by Delta. Molecularly, interactions might occur at the protein level because rugose appears not to be a direct transcriptional target of Notch (Schreiber, 2002).

echinoid (ed) encodes a cell-adhesion molecule (CAM) that contains immunoglobulin domains and regulates the Egfr signaling pathway during Drosophila eye development. Genetic mosaic and epistatic analysis, has suggested that Ed, via homotypic interactions, activates a novel, as yet unknown pathway that antagonizes Egfr signaling. Alternatively, later studies indicate that Ed inhibits Egfr through direct interactions. Another body of work suggests that Ed functions as a homophilic adhesion molecule, and also engages in a heterophilic trans-interaction with Drosophila Neuroglian (Nrg), an L1-type CAM. Co-expression of ed and nrg in the eye exhibits a strong genetic synergy in inhibiting Egfr signaling. This synergistic effect requires the intracellular domain of Ed, but not that of Nrg. A model for this interaction suggest that Nrg acts as a heterophilic ligand and activator of Ed, which in turn antagonizes Egfr signaling (Spencer, 2003 and references therein; Islam, 2003 and references therein).

Complicating the picture even further is an analysis of a paralogue of Ed termed friend of echinoid (fred). ed and fred transcription units are adjacent to one another, approximately 100 kilobases apart on chromosome arm 2L, but they are divergently transcribed in opposite directions. Fred acts in close concert with the Notch signaling pathway. Suppression of fred function results in specification of ectopic SOPs in the wing disc and a rough eye phenotype. Overexpression of N, Su(H), and E(spl)m7 suppresses the fred RNAi phenotypes. Accordingly, decreasing Su(H) or overexpression of Hairless enhances the fred RNAi phenotypes. Thus fred, a paralogue of ed, shows close genetic interaction with the Notch signaling pathway. The weak genetic interaction observed between fred and components of the Egfr pathway also links fred to the Egfr pathway; however, analysis of additional components of the Egfr pathway are necessary to determine Fred's role in the Egfr signaling (Chandra, 2003).

In order to study the function of fred, the heritable and inducible double-stranded RNA-mediated interference (RNAi) method was used. For this study, transcript sequence of fred was cloned as a dyad symmetric molecule in the pUAST vector and transgenic lines established. Expression of the construct was induced by crossing the transgenic lines to tissue- and/or stage-specific GAL4 driver lines. Transcription of a dyad symmetric molecule results in a RNA that snaps back to give rise to a dsRNA with a hairpin loop; this mediates the degradation of the corresponding endogenous mRNA. A 638-bp region of fred was selected for this analysis based on minimal similarity to ed sequence (Chandra, 2003).

The Notch signaling pathway is involved in limiting the SOP fate to a single cell within each proneural cluster. Since degradation of fred mRNA leads to formation of ectopic SOPs, it was of interest to see if the Notch signaling pathway genes functionally interact with fred in this process and, thus, may modulate the fred RNAi phenotype. To this end, four Notch pathway genes, Notch (N), Suppressor of Hairless [Su(H)], Hairless (H), and E (spl) m7 were tested for genetic interactions with fred (Chandra, 2003).

H antagonizes Notch target gene activation by binding to the Notch signal transducer, Su(H). Accordingly, overexpression of H phenocopies reduction of Notch activity. Ectopic expression of H in the pnr domain results in the formation of multiple/split bristles and loss of epidermal tissue. This phenotype is enhanced in animals with suppressed fred activity in the pnr domain. Functional interactions between H and fred are also evident in the eye. UAS-H/GMR-GAL4 flies have eyes that are slightly smaller along the anterior-posterior axis and show ommatidial fusion and interommatidial bristle tufting, as well as bristle loss. When fred activity is suppressed in this genetic background, there is an enhanced disruption of the eye morphology. Ommatidia lack definition, bristle tufting is more severe, and loss of bristles is observed (Chandra, 2003).

The observations that changes in the activity of four genes of the Notch signaling pathway can either suppress or enhance the phenotypes associated with the suppression of fred function suggest that fred is functioning in close concert with the Notch signaling pathway. Reduction in the activity of a Notch signaling pathway gene, Su(H) results in an enhancement of the fred RNAi phenotype. In contrast, ectopic expression of Notch signaling pathway genes, Notch, Su(H), and E(spl)m7 suppresses, to different degrees, different aspects of the fred RNAi phenotype in the developing wing, thorax, and eye. In contrast, overexpression of Hairless (a negative regulator of the Notch pathway) enhances the phenotypes induced by Fred suppression. It is presently not clear whether Fred defines a separate pathway for SOP determination or if it shares downstream components of the Notch signaling pathway. The remarkable degree to which ectopic expression of an E(spl) complex bHLH transcription factor results in a nearly complete suppression of phenotypes associated with fred degradation strongly supports the idea of very close functional interactions. These observations, furthermore, raise the possibility that E(spl) complex genes and/or other genes of the Notch signaling pathway act downstream of fred function (Chandra, 2003).

Hairless interaction with the Egf pathway

The Drosophila epidermal growth factor receptor (Egfr) is a key component of a complex signaling pathway that participates in multiple developmental processes. Carried out was an F1 screen for mutations that cause dominant enhancement of wing vein phenotypes associated with mutations in Egfr. In this screen, mutations in Hairless (H), vein, groucho (gro), and three apparently novel loci were all recovered. All of the dominant enhancer mutations, termed E(Egfr)s, identified show dominant interactions in transheterozygous combinations with one another and with alleles of N or Su(H), suggesting that they are involved in cross-talk between the N and Egfr signaling pathways. Further examination of the phenotypic interactions between Egfr, H, and gro reveal that reductions in Egfr activity enhance both the bristle loss associated with H mutations, and the bristle hyperplasia and ocellar hypertrophy associated with gro mutations. Double mutant combinations of Egfr and gro hypomorphic alleles lead to the formation of ectopic compound eyes in a dosage sensitive manner. These findings suggest that these E(Egfr)s represent links between the Egfr and Notch signaling pathways, and that Egfr activity can either promote or suppress Notch signaling, depending on its developmental context (Price, 1997).

Wing vein development in Drosophila is controlled by different morphogenetic pathways, including Notch. Hairless (H) antagonizes Notch target gene activation by binding to the Notch signal transducer Suppressor of Hairless [Su(H)]. Accordingly, overexpression of H phenocopies reduction of Notch activity. In the construct H-C2, the presumptive Su(H)-binding domain of Hairless has been removed. As a consequence, H-C2 protein has completely lost the ability to bind to Su(H) protein and to interfere with Su(H)-dependent developmental processes in vivo like bristle development, wing margin specification or vein width refinement. Apart from the internal deletion, the H-C2 protein compares to the wild type with respect to antibody recognition, apparent molecular weight, subcellular distribution, and stability as well as biochemical interactions with other H partner proteins. With regard to endogenous activity, the H-C2 lines are rather weak, compared to the full length H constructs; however, after heat shock induction, expressivity is similar. Surprisingly, overexpression of H-C2 induces lethality as does the wild type construct and in addition, leads to the induction of ectopic vein material in certain intervein regions of the wing (Johnnes, 2002).

Keeping in mind that H itself is not a transcriptional regulator but rather functions through protein-protein interactions with different protein targets, these phenotypes cannot be simply explained by altered activation of Notch target genes. Rather, they might uncover a currently unknown Su(H) independent activity of H involving different protein(s) maybe outside of the Notch signaling cascade. In order to understand this phenomenon, the involvement of H-C2 in the process of vein establishment was analyzed in comparison to wild type H. The data presented in this work are in agreement with a model whereby H, apart from antagonizing Notch signaling, positively regulates EGF signaling during the process of wing vein formation (Johnnes, 2002).

In a screen for genetic modifiers of the H-C2 phenotype, several genes involved in Notch and epidermal growth factor (EGF) signaling were identified. Most notably veinlet (rhomboid), an activator of EGF signaling, acts downstream of H-C2. H-C2 positively regulates veinlet maybe through inhibition of intervein determinants in agreement with a model, whereby Notch and EGF signaling pathways cross-regulate vein pre-patterning (Johnnes, 2002).

Overexpression of hs-H-C2 induces ectopic veins only between day 5 and 6 after egg deposition. Phenotypes vary significantly and were arranged into a phenotypic series of five classes. Using precisely synchronized cultures, the pheno-critical period was restricted to pre-pupal and early pupal developmental stage, starting approximately at the larval-prepupal transition. Induction of H-C2 during mid- to late-third instar larval stages did not result in ectopic vein formation even with prolonged and, in their consequence, semi-lethal heat shocks. Ectopic venation was not randomly distributed and certain regions of the wing blade were more sensitive than others. In order to distinguish between temporal and/or sensitivity differences, the wing was partitioned into distinct intervein sectors A-F and the appearance of extra veinlets over time for each sector was scored independently. As became apparent, the six different sectors respond with a similar temporal profile, but with different sensitivities. The less sensitive sectors A and C revealed two pheno-critical periods, which might, however, be a consequence of the time convolution of the data. In summary, the main impact of H-C2 on the wing venation process occurs in the pre-pupal and early pupal stages of development (Johnnes, 2002).

The H-C2 protein is unable to bind to Su(H) and has lost basically all of H wild type activity: H-C2 is unable to rescue the haplo-insufficient H loss-of-function phenotype and does not cause the typical H gain-of-function bristle phenotypes. Thus, H-C2 venation phenotypes might either uncover a Su(H) independent function of H or an unrelated, novel activity. Although overexpression of H wild type constructs causes only little ectopic vein material, H and H-C2 proteins are able to synergize upon overexpression. It is concluded that the vein inductive property of H-C2 is a native function of H, and that H is able to partially substitute for H-C2 in this process (Johnnes, 2002).

If indeed the vein inductive property of H-C2 uncovers a Su(H) independent activity of H, the question arises as to what other targets H might act upon. In order to identify such putative targets, a candidate screen was set up for dominant modifiers, concentrating on the three main signaling pathways which normally contribute to wing vein development: Notch, Egf and Dpp signaling cascades. In the double heterozygotes, H-C2 was induced during the pheno-critical period with double heat shocks to make up for the weak phenotypes caused by a single hs-H-C2 copy (Johnnes, 2002).

In a first set of experiments, combinations with Notch-family members and relatives were analyzed. These included mutations in the Notch receptor itself and in Notch ligands, mutations affecting the signal transduction machinery as well as Notch target genes. Furthermore, other neurogenic mutations as well as several proneural members were included in the screen. Reduction of single doses of Dl or components of the E(spl) complex including E(spl)mß, enhance the H-C2 wing phenotype in a dominant manner. The behavior of most of the other components of the Notch signaling pathway were largely neutral in this screen. Interestingly, N loss of function alleles act as strong negative modifiers, whereas N^Ax alleles enhance the response to H-C2 overexpression (Johnnes, 2002).

Components of the EGF signaling pathway, screened for dominant interactions with H-C2, included ligand and its processing, the receptor itself, components of the signal transduction cascade as well as related genes. As expected, ve and to a lesser degree Star (S) mutations dominantly reduce the amount of extra vein material. Both genes are essential for vein formation, and thus, reduction of their activity was expected to antagonize the H-C2 vein-promoting effect. By lowering the ve gene dose, the suppression is almost complete except for some ectopic vein material in the region of the anterior cross-vein, a region which is not affected by the homozygous ve¹. Only weak, interactions were found with Dpp pathway mutations (Johnnes, 2002).

In agreement with the essential role of ve in the establishment of vein fate, data indicated that the homozygous ve¹ mutant completely suppresses ectopic vein induction through hs-H-C2, except for some small veinlets. No ectopic veins were visible in the distal wing blade, where the ve¹ phenotype is apparent, even after strong overexpression. This demonstrates that H-C2 strictly depends on ve for the induction of veins and suggests that overexpression of H-C2 might somehow result in the ectopic activation of ve (Johnnes, 2002).

In accordance with the proposed role of net as negative regulator of ve, net¹ mutations cause extensive extra veins. This phenotype comprises nearly all aspects of hs-H-C2 overexpression in a wild type background. Unexpectedly, overexpression of H-C2 enhances the net phenotype considerably: not only do the heterozygous net¹ mutants resemble the homozygotes, but the homozygotes developed massive patches of vein tissue and extensive blistering of the wing. Moreover, net¹; hs-H-C2 homozygotes were semi-lethal at 25°C and the stock was only viable at 18°C. Because net¹ is a complete null allele, this result excludes the simple model that H-C2 promotes vein development by inhibition of net activity. Instead, H-C2 acts independent of net either as a vein-promoting factor, e.g., by activation of ve, or by repression of other negative regulators of ve that act in addition to net. The latter seems more plausible with regard to normal H function. In the heterozygous net¹ background, which is phenotypically wild type but sensitized for ectopic vein formation, the vein-promoting activity of H is revealed: overexpression of full length H in net¹ heterozygotes results in ectopic venation that is a perfect phenocopy of the H-C2 effects. Apparently, wild type H has a vein-promoting activity, which also can be explained by antagonizing a negative regulator of ve (Johnnes, 2002).

Since induction of ectopic vein material by H-C2 is extremely sensitive to developmental time, the epistatic relationship with ve¹ was reassessed by continuously overexpressing H-C2 or H with the aid of the Gal4/UAS system in a wild type and a ve¹ background. Prolonged overexpression of H-C2 with en-Gal4 or Bx^MS1096-Gal4 driver lines results in conversion of most of the distal intervein areas to vein tissue. Only the region between L3 and L4 proved resistant. Furthermore, induction of microchaetae in this area was observed. Apart from this conversion, extensive tissue loss was noted while the wing margin itself remained intact. Overexpression of H-C2 in the ve¹ background shows both tissue loss and ectopic microchaetae on the wing blade. Again, no induction of ectopic vein material was observed in the more distal regions in support of the notion that H-C2 acts upstream of ve. It is proposed that H-C2 promotes vein induction by up-regulating ve activity. Moreover, processes independent of ve are influenced by H-C2, which finally lead to the induction of ectopic bristles on the wing blade and to tissue loss. Overexpression of H in the same experimental set-up was unable to convert intervein tissue into vein material, apart from few ectopic veinlets. At the same time, H causes wing tissue and margin loss accompanied by broadened wing veins. These are the known hallmarks of an impaired Notch signaling (Johnnes, 2002).

Since vein determination depends on the balance between ve and bs activity, the influence of H or H-C2 on wild type bs or ve gene expression was examined. Full length H and H-C2 expression was ectopically induced from UAS-constructs in the posterior compartment via the en-Gal4 driver. ve expression was monitored with either enhancer trap line, ve^rho-lac1 or ve^X81. Expression of ve and bs is mutually exclusive in vein and intervein tissue, respectively. Overexpression of H does not alter this complementary expression pattern, however, overexpression does cause strong ve expression within and confined to pro-vein areas. This is in contrast to the effects of H-C2 overexpression where ectopic expression of ve^rho-lac1 is very prominent in pupal wings already in early pupal stages and remains strongly activated at least until 36 h after pupal formation (APF). Earlier wing development was analyzed using the ve^X81-lacZ reporter. In wing discs from late third instar larvae, no patterning defects apart from tissue loss are observed. However, deviations from wild type become apparent already in the pre-pupal wing discs as early as 4 h APF. At 6 h APF, a strong and reliable ectopic staining near the wing margin, where pro-vein 5 develops, is observed that later spreads into the adjacent intervein field. Overall, ectopicve expression reliably predicts the pattern of ectopic venation caused by overexpression of H-C2. This is in agreement with the hypothesis that ve regulation is a target of H-C2 activity (Johnnes, 2002).

The simplest model to explain the differential effects of H and H-C2 on the regulation of ve expression would be the assumption of a vein inductive role of Notch in pre-pupal and early pupal development in agreement with the biphasic developmental role of Notch, e.g., during eye development, where, in the course of lateral inhibition, Notch first promotes proneural fate before restricting this fate to single photoreceptor precursor cells. By binding to Su(H), H would limit such a vein-promoting activity of Notch at an early inductive phase. Because H-C2 is unable to bind to Su(H), an assumed inductive Notch signal would be able to pass and thus, set the stage for ectopic veins. To test this assumption, an activated Notch receptor (N^intra) was expressed under heat shock control during the H-C2 pheno-critical period. Since hs-N^intra overexpression at 37° proved extremely lethal to larvae and pupae alike, the induction was performed at lower temperatures of 34-35°. Under these conditions, hs-N^intra is able to induce ectopic veinlets, mostly of cross-vein character, in all the regions where H-C2 is also able to induce ectopic vein material. These results demonstrate that Notch signaling is able to exert a positive influence on wing vein specification during early pupal stages, closely followed by the well characterized vein suppressing activity of Notch signaling (Johnnes, 2002).

The onset of pupariation is a major developmental switch, where expression of many genes as well as their developmental effects changes dramatically. This is, for example, observed in the regulation of ve and bs from third instar larval stage to early pupal stage: whereas the activation of both genes early on depends on pre-pattern genes like net, their regulation becomes inter-dependent and mutually exclusive about 4 h after puparium formation. The abrupt onset of H-C2 vein-promoting activity is interpreted accordingly. Maybe, H-C2 responds to or influences the activity of other factors which only become available at that time and play a role in the promotion or repression of vein development. This is reflected by the onset of the positive influence of H-C2 on ve expression at around 4 h after pupariation. Thus, the H-C2 pheno-critical period might reflect a developmental switch for a requirement of H activity for vein fate decisions (Johnnes, 2002).

Involvement of the Notch signaling pathway in the refinement of proper vein width is well established. Current models suggest that during this lateral inhibition process, Dl acts as an inhibitor of vein formation by directing cells, neighboring presumptive vein cells, into the intervein fate. This model is in line with the observation that Dl mutants act as enhancers of H-C2 vein promotion. In Dl mutants, the threshold for vein fate is lowered as determined vein cells are less likely to be driven back into intervein fate. The interrelationship of H-C2 and Notch signaling during vein formation is, however, not restricted to the process of vein width refinement. Overexpression of N^intra promotes early vein formation and may thus be setting the stage for pro-vein development within intervein areas. Despite the fact that Notch activity is not necessary for pro-vein specification itself, it is required for the vein-promoting activity of H-C2 because Notch mutations act as strong dominant suppressors of H-C2 effects. In agreement, N^Ax-E2, a hyper-activated allele of Notch, acts as a weak enhancer of H-C2 and it is even possible that this effect is initially stronger but then obliterated through enhanced lateral inhibition (Johnnes, 2002).

Reduction of the ve gene dose results in a very pronounced, dominant repression of the H-C2 phenotype despite the fact that the allele ve¹ has no dominant visible phenotype. This suggests that ve plays a crucial role for H-C2 to exert its inductive effects. Interestingly, dosage reduction of either the Drosophila EGF receptor or the MAPK rolled (rl) has no dominant influence on the H-C2 ectopic venation phenotype. The former result was unexpected and suggests that the Drosophila EGF-receptor itself is not rate limiting in this process. This notion is in line with the observation that also ve¹ is fully recessive in combination with loss-of-function alleles for the Drosophila EGF-receptor. The allele rl¹ is a mild hypomorph and the reduction of MAPK activity might not be strong enough to influence the H-C2 phenotype. Together with the results of full epistasis of ve over H-C2, these data suggest that neither the Drosophila EGF-receptor nor the EGF signal transduction cascade are influenced by overexpression of H-C2 (Johnnes, 2002).

Overexpression of H results in the extension of ve expression all over the pro-vein area, whereas overexpression of H-C2 induces, in addition, ve outside the pro-veins, also within the intervein fields. Thus, both act positively on ve regulation but H-C2 is clearly different from H with respect to the apparent conversion of presumptive intervein- to pro-vein cell fates. Pro-vein activity is a normal aspect of H wild type function that is uncovered in a sensitized background: halving the gene dose of net or bs might result in a subtle increase of ve activity which can then be pushed by H above the threshold for pro-vein fate. Although the results clearly demonstrate that H and H-C2 act positively on the regulation of ve, it cannot be concluded that this regulation is direct. Rather H might act negatively on the output of vein repressing factors. Since H has the capability to interact with a number of different proteins, overexpression of either H or H-C2 could influence stoichiometry of complexes or availability of factors involved in ve regulation. Two such factors that have overlapping activity with regard to the negative regulation of ve are encoded by net and bs, however, they show remarkably different temporal activity profiles in that net acts during larval stage and bs at the transition of larval to pre-pupal stage. Thus, bs appears the more likely target of H activity; this is supported by the fact that H-C2 can promote vein induction even in the absence of net and that bs repression is observed at the anterior cross-vein without simultaneous ve induction. Whether this inhibition is direct or via as yet unknown factors and pathways requires further study (Johnnes, 2002).

Thus, both H and H-C2 have the ability to up-regulate ve. However, in contrast to H, H-C2 overexpression is capable of overriding intervein fate and thus inducing ve expression within intervein territories. Several lines of evidence suggest that this is a normal facet of H function. (1) Synergistic effects of combined overexpression of H and H-C2 were found. (2) In a sensitized genetic background of reduced copies of intervein specifying genes like bs and net, H itself possesses vein-promoting activity just like H-C2. These results can be explained if one assumes a dual, independent activity for H: in one case, H might up-regulate ve, for example, by interfering with intervein specifying factors, an activity retained by H-C2. In another case, H, by virtue of binding to Su(H), antagonizes Notch-dependent processes such as an early vein fate-promoting activity and subsequently vein width refinement. This activity is presumably lost in H-C2 due to the lack of the Su(H)-binding domain. Unfortunately, attempts to directly test this hypothesis failed because Su(H) mutant clones could not be generated in the background of H-C2 (Johnnes, 2002).

A dual role of H in suppressing inductive Notch signaling and enhancing ve activity would explain why the wild type protein is unable to induce ectopic venation except in a sensitized background, where ve activity has already reached a critical threshold by the reduction of its negative regulators. In contrast, H-C2 can no longer antagonize Notch activity, but might still promote vein formation by interfering with ve suppression. Altogether, the genetic data may be taken as an example for a link between Notch and EGF signaling, which in the context of vein formation appears to involve the activity of H influencing both Notch signaling and, via ve, EGF signaling as well (Johnnes, 2002).

Genetic modifier screens on Hairless gain-of-function phenotypes reveal genes involved in cell differentiation, cell growth and apoptosis in Drosophila melanogaster

Overexpression of Hairless (H) causes a remarkable degree of tissue loss and apoptosis during imaginal development. H functions as antagonist in the Notch (N) signaling pathway in Drosophila, and the link to growth and apoptosis is poorly understood. To gain further insight into H-mediated apoptosis, two large scale screens were performed for modifiers of a small rough eye phenotype caused by H overexpression. Both, loss- as well as gain-of-function screens revealed known and new genetic interactors representing diverse cellular functions. Many of them do not cause eye phenotypes on their own, emphasizing a specific genetic interaction with H. As expected, the components of different signaling pathways identified were supposed to be involved in the regulation of cell growth and cell death. Accordingly, some of them also act as modifiers of proapoptotic genes, suggesting a more general involvement in the regulation of apoptosis. Overall, these screens highlight the importance of H and the N pathway in mediating cell death in response to developmental and environmental cues and emphasize their role in maintaining developmental cellular homeostasis (Muller, 2005).

Programmed cell death is used to remove damaged or supernumerary cells and serves as a substantial patterning mechanism during the development of complex animal structures. In Drosophila, apoptosis has been shown to be required, e. g., for shaping of the nervous system, patterning of the pupal eye, metamorphosis or proper development of germ cells. Crosstalk between different signaling pathways fuels differentiation and apoptosis alike. The N signaling pathway is one example of a cell-cell communication pathway involved in a large number of cell fate decisions that is associated with apoptotic processes as well. This study aimed at finding factors that modify apoptotic phenotypes resulting from overexpression of H in the eye. Both a misexpression and a loss-of-function screen were performed based on chromosomal deficiencies. This twofold approach allowed the strengths of one to be played off the weaknesses of the other. While a deficiency-based screen can quickly map loci interacting with H, it can be difficult to subsequently identify specific mutations that account for this interaction. In addition, since only a fraction of mutations results in visible phenotypes, modifiers may go unnoticed especially in cases of gene duplication and redundancy. Therefore, a complementary overexpression screen may identify genes that are missed otherwise. In the past, gain-of-function genetics has been successful in identifying genes crucial to different developmental processes like oogenesis, tissue growth, sensory organ development or thorax formation. The gain-of-function screen identified a total of 86 factors including 57 enhancers and 29 suppressors. Effects arising from the misexpression of these factors on their own are a potential drawback of this screen: roughly 40% of the enhancers and 60% of the suppressors displayed phenotypes on their own when overexpressed in the eye. However, more than 50% of them (44 out of 86) showed the opposite effect on GMR (a glass promoter construct) driven H when tested in the respective loss-of-function mutant background, arguing for a specific connection to H. Moreover, some of the genes identified in this screen may regulate the glass gene itself. This was taken into account by testing all identified suppressors for their rescue ability of tissue loss and apoptosis caused by H overexpression during wing development. In support of specific interactions with H, the majority (23 of 29) ameliorated these effects arguing against an involvement of glass (Muller, 2005).

In the loss-of-function screen, 41 deficiencies were recovered and 36 different loci were subsequently mapped, 22 acting as suppressor and 14 as enhancer. 10 of them were also recovered in the gain-of-function screen. One explanation might be, that altogether the deficiencies uncovered just 75% of the genome, leaving a quarter uninspected. Moreover, the collection of EP lines used accounts for roughly 10% of the genes in the entire genome. These numbers illustrate the benefit of taking various genetic approaches and emphasize that no single screen will identify all or even most potential interactors (Muller, 2005).

The N signaling pathway regulates a plethora of developmental processes including various differentiation steps and cell death during eye development. Since H acts as general antagonist of N, one might expect a variety of diverse factors to modify phenotypes caused by H overexpression. For this reason, the isolated modifiers were subjected to further analyses with regard to their own phenotypes and their general involvement in apoptosis (Muller, 2005).

A majority of the 57 enhancers [33 or 58%] caused no phenotype or even bigger eyes upon overexpression, indicating that they do not induce tissue loss on their own. Interestingly, 15 of them were also identified in screens conducted to find factors involved in thorax formation, bristle development, mesoderm development, cell growth in the eye or synapse formation. Since N signaling regulates various aspects in the development of these different tissues and organs, one might speculate that this group of enhancers affects N activity primarily during differentiation processes. Although not identified in the other screens, the remaining 14 factors, belonging to functional categories as diverse as growth regulators, transcription factors or protein kinases and enzymes might be connected to the N signaling pathway as well, thus reflecting the manifold N dependent processes in the development of Drosophila (Muller, 2005 and references therein).

A total of 24 factors showed no apparent effect in cell death assays. They comprise several N pathway components (emc, sd, tom), the novel factor CG8788 and also smrter, which functions as a co-repressor and might mediate transcriptional repression of N target genes also in Drosophila. However, most of the genes in this category have functions related to cell division and cell growth. For example, Dap overexpression reduces growth and proliferation in the eye imaginal disc and causes lethality upon combined misexpression with H. Consistent with the notion that levels of dMyc determine growth and cell proliferation, mutants in this gene are also lethal in trans with GMR driven Hairlesws. dMyc activity is regulated by several morphogens and the results suggest that N may also be involved at least for some aspects of dMyc regulation. Another group of genes, including bazooka, fat, inflated or Rok, functions in cell adhesion and cell polarity. The incorrect establishment of epithelial polarity is accompanied by hyperplastic growth which can be synergistically enlarged by an ectopic N signal. The finding that mutations in any of these loci behave as suppressors of H overexpression raises the possibility of a rather direct connection to the N signaling pathway. Thus, this screen uncovered several genes, which influence H and N activity during growth and proliferation, raising the question of their molecular role in the N signaling pathway (Muller, 2005).

Exactly 50% of all different modifiers (56 of 112) were either rescued by DIAP1 or influenced cell death inducers themselves. The recovery of factors known to be generally involved in apoptosis, like reaper, thread and bantam or more specifically during eye development like klumpfuss, was not only expected but was demanded by the screening approach. Interestingly, two of these genes (Rac2 and Eip78C) were in a data set collected in course of a genome-wide analysis of steroid-triggered cell death response in Drosophila. A further connection between N and the ecdysone regulatory network was recently established during metamorphosis of the midgut. The regulatory input of EGFR-signaling as well as crosstalk with N-signaling in the control of cell death has been shown at different stages of Drosophila development, most notably in the eye. In agreement with these earlier findings, several EGFR-pathway members were identified as modifiers of H and cell death inducers alike (e.g. aop, DER, lilli, pnt and rho). More interestingly, several members were identified of the JNK pathway (e.g. bsk, hep, msn, puc), which has been involved earlier in morphogenetic as well as stress induced apoptosis. Genetic analyses have demonstrated that JNK signaling is an effector of larval and pupal apoptosis. During embryogenesis N signaling has a negative effect on JNK signaling in the process of dorsal closure. However, this seems to involve non-canonical N signaling. Besides this influence on patterning processes, this work points to an involvement of canonical N signaling in JNK mediated morphological cell death. In this context it is interesting to note that the screen also identified a phosphatase subunit: overexpression of PP2A-beta' (B56) encoded by widerborst strongly suppresses H, p53 and grim induced cell death in the eye, whereas wdb mutants act as enhancers of H. In agreement, knockdown of B56 PP2A during embryogenesis results in caspase activation. Genetically, it has been placed in the p53-regulated path of apoptosis (Muller, 2005).

A strong correlation was found between H induced cell death and p53 mediated apoptosis. For example, cell death induced by overexpression of p53 can be rescued by increasing N signals [N- or Su(H) gain- or H-loss-of-function]. Further studies will determine the molecular mechanisms underlying these genetic interactions (Muller, 2005).

Many of the identified interactors have been previously implicated in different aspects of development but not in apoptotic processes. One example is the IMP (IGF-II mRNA-binding protein). IMP is ecdysone inducible and was suggested to be involved in the regulation of translation, maybe during metamorphosis, arguing for a role of IMP in regulating cell death in this context. Interestingly, several genes involved in chromatin remodeling were identified as strong interactors of H and the other tested cell death inducers. The Drosophila Brahma complex plays an important role during G1 phase of the cell cycle. In the current study, brm2 mutants behaved as enhancer of H and the proapoptotic genes hid, rpr and grim, but not of the stress-induced p53 apoptotic pathway. This argues for an additional role of brm in the coordination of cell death, besides its well defined function in the regulation of cell growth. Another example is DspI that was identified in these screens as general repressor of apoptosis. DspI encodes a transcriptional corepressor that binds to Dorsal and Relish (Rel) proteins. Like their mammalian counterparts, Rel proteins mediate immune-response via JNK signaling. It is tempting to speculate that Rel proteins together with DspI might likewise protect against apoptosis by limiting the JNK signal. In this case, the effects of Dsp1 on H mediated apoptosis can be easily explained and provide a further link for a crosstalk between JNK and N pathway. A third factor in this group is the Drosophila CREB binding protein, encoded by the nej locus, and belonging to the CBP/p300 family. nej is required at successive stages of eye development and overexpression caused severe retina degeneration. Since nej mutants have anti-apoptotic effects on H and most cell death inducers alike, one might assume a 'manager' function in the control of cellular homeostasis and apoptosis (Muller, 2005).

Last, seven of 15 interactors with hitherto unknown function were shown to interfere with apoptosis. The future challenge will be to determine the molecular and functional relationship between these new genes and cell death induction by H (Muller, 2005).

The screens provided a wealth of new information regarding cell death induction observed after overexpression of H. The results are compatible with the notion that changes in N activity have an effect on cell death in response to abnormal or imbalanced developmental signals within a cell. In agreement, the identified modifiers include factors and signaling components like p53, JNK-signaling and hormone triggered factors, all known to be involved in the coordination of a wide range of biological responses, including growth, differentiation and programmed cell death. Apparently, N signaling is required for the correct interpretation of such developmental signals and for the crosstalk between different signaling pathways that is essential for cell survival and differentiation (Muller, 2005).

putzig is required for cell proliferation and regulates notch activity in Drosophila

The gene putzig (pzg) is a key regulator of cell proliferation and of Notch signaling in Drosophila. pzg encodes a Zn-finger protein that exists within a macromolecular complex, including TATA-binding protein-related factor 2 (TRF2)/DNA replication-related element factor (DREF). This complex is involved in core promoter selection, where DREF functions as a transcriptional activator of replication-related genes. This study provides in vivo evidence that pzg is required for the expression of cell cycle and replication-related genes, and hence for normal developmental growth. Independent of its role in the TRF2/DREF complex, pzg acts as a positive regulator of Notch signaling that may occur by chromatin activation. Down-regulation of pzg activity inhibits Notch target gene activation, whereas Hedgehog (Hh) signal transduction and growth regulation are unaffected. These findings uncover different modes of operation of pzg during imaginal development of Drosophila, and they provide a novel mechanism of Notch regulation (Kugler, 2007; full text of article).

In a developing organism, cell proliferation and apoptosis must be strictly coordinated with patterning processes to correctly shape the organs. Thus, it is not surprising that all major morphogenetic and developmental signaling pathways have been involved in the regulation of cell proliferation and apoptosis and that they have been linked to numerous cases of cancer formation in mammals. In Drosophila, a large body of work shows that several of these pathways act in concert in the coordination of cell survival and death. For example, overexpression of Notch causes vast overgrowth, whereas inhibition of Notch activity by overexpression of its antagonist Hairless results in tissue loss and apoptosis. Indeed, the combined activity of Hedgehog (Hh), Decapentaplegic (Dpp), and Notch is required to promote reentry into the cell cycle after a developmentally regulated G1 arrest in the eye anlagen of Drosophila larvae. Moreover, it was shown that Hh signaling promotes cellular growth by transcriptional activation of G1 cyclins Cyclin D and Cyclin E. However, to this end, the understanding of the molecular principles that connect these pathways to either control of cell cycle or apoptosis remains largely fragmentary (Kugler, 2007).

Cell cycle entry requires the activity of G1-S cyclins that eventually activate dE2F1, a transcription factor that induces transcription of downstream genes required, e.g., for DNA replication. In Drosophila, transcriptional activation of replication-related genes encoding, for example, proliferating cell nuclear antigen (PCNA) or DNA-polymerase alpha subunit involves also DNA replication-related element factor (DREF) that recognizes DNA replication-related element (DRE) response elements. DREF can be part of a macromolecular complex including TRF2, a TATA-binding protein-related factor that binds to a subset of selected promoters, including one promoter in the PCNA gene. TRF2 has been isolated from several different organisms, where it is required for transcription of replication-related genes and key developmental genes as well. The TRF2/DREF complex consists of more than a dozen proteins, including several known chromatin-remodeling components. Three of them confer chromatin activation, whereas two others, including p160, resemble regulators of insulator function. Interestingly, p160 was recently found to enhance position effect variegation and hence chromatin silencing and to be associated with interband regions of polytene chromosomes (Eggert, 2004). To this end, the biochemical activity and functional specificity of most of the proteins within the TRF2-complex, i.e., their role in transcriptional activation or in chromatin remodeling, however, remain elusive (Kugler, 2007).

This study isolated the Zn-finger protein p160 as a genetic suppressor of Hairless activity, prompting an interest in its role during Drosophila development and especially during Notch signaling. In vivo RNA interference resulted in tiny larvae and developmental delay, which is why the corresponding gene was named putzig (pzg). This study presents in vivo evidence that pzg is essential for fly survival by regulating cell cycle entry and progression. In addition, pzg encodes a key regulator of the Notch signaling pathway and that it is involved in histone modification and chromatin activation. Interestingly, this activity is independent of DREF, suggesting context dependence of Pzg activity (Kugler, 2007).

EP756 was identified in a genetic screen as suppressor of tissue loss caused by an overexpression of the Notch antagonist Hairless (H) during eye development. This positive effect was not restricted to the eye, because it was likewise observed during wing development. Moreover, cell growth and proliferation induced by an enforced Notch signal was significantly enhanced (~20%) by a combined overexpression with EP756. Tissue specific overexpression of EP756 caused a very mild enlargement of the respective tissues on its own. These data suggest a more general role of EP756 in the control of cell proliferation as well as an intimate interaction with Notch signaling (Kugler, 2007).

Pzg is one component of a multiprotein complex that contains TRF2 and DREF. TRF2 allows transcription initiation from selected promoters independently of TFIID. DREF is a positive transcriptional regulator of cell cycle and replication-related genes, and it may guide TRF2 to the PCNA and DNA-polymerase alpha promoters. Assuming promoter recognition or binding requires Pzg contained within the TRF2/DREF complex, depletion of Pzg might destroy the complex or reduce its activity, easily explaining the dramatic proliferation defects. However, it is noted that only a subset of promoters containing DREF binding sites involves activation through TRF2, suggesting that DREF can act independently of TRF2. Moreover, Pzg activity is found independently of DREF, indicating that TRF2/DREF complex components can act either alone or in conjunction with other factors (Kugler, 2007).

The TRF2/DREF complex contains several proteins involved in chromatin remodeling. Notably, Pzg and one other TRF2/DREF component p190 are reminiscent of factors implicated in insulator function. In accordance, Pzg activity has been associated with position effect variegation and chromatin silencing. In contrast, assays reveal an essential function of Pzg in retaining robust K4-trimethylation of histone H3, which is directly associated with open chromatin structures. In accordance with these findings, EP756 was recently identified as a suppressor of the cut allele ct^K. This cut mutation is caused by the insulator activity of a gypsy retrotransposon, which can be relieved by EP756 overexpression. EP756 is shown in this stody to drive Pzg expression, in support of the notion that Pzg's epigenetic activity overcomes gypsy insulator function (Kugler, 2007).

Three of the proteins found in the TRF2/DREF complex have been identified previously in the nucleosome-remodeling factor NURF (see NURF301), which consists in total of four subunits. NURF is associated with chromatin activation by facilitating transcription of chromatin in vivo. In fact, mutations in Drosophila ISWI, the catalytic subunit of NURF, and other nucleosome remodeling complexes caused phenotypes that are very reminiscent of pzg-RNAi-induced defects. Because DREF down-regulation has no effect on trimethylation of H3K4, it seems unlikely that the TRF2/DREF complex as a whole is involved in chromatin activation. Instead, Pzg may be part of a NURF-like chromatin-remodeling complex, depending on the developmental context (Kugler, 2007).

Apart from a role in proliferation, this study has uncovered an important role for Pzg as positive regulator of Notch signaling. Interestingly, it was found that Pzg binds to chromatin in the regulatory region of the Notch target genes E(spl)m8 and vg. This regulation is independent of DREF: albeit DREF binding sites are common to Drosophila promoters, neither Notch nor Notch target genes that were investigated are transcriptional targets of DREF. Thus, reduced transcriptional activity of Notch target genes in pzg-RNAi mutant cells is due to a DREF-independent role of Pzg. Alternatively, Pzg could facilitate formation of the transcriptional activator complex that is assembled on Notch target promoters involving intracellular Notch itself. By using the yeast two-hybrid system, several Notch pathway members were tested; however, no binding to Pzg was detected. It is proposed that Pzg has a dual function that is effected differently. On one hand, it activates proliferation-related genes in conjunction with TRF2/DREF, and on the other hand, it activates Notch signaling by chromatin activation independently of DREF (Kugler, 2007).

Several lines of evidence support the idea that Notch signaling is particularly susceptible to chromatin remodeling. For example, Notch transcriptional activity requires the histone-modifying enzyme dBre1 that is indirectly required for K4-methylation of histone H3. Moreover, chromatin-modifiers were also shown to potentiate Notch activity during Drosophila wing development. Finally, general transcriptional regulators and chromatin remodeling factors were found in several independent genetic screens to influence Notch signaling, indicating to a role of pzg in linking Notch to chromatin remodeling. The bimodal activity of Pzg onto both cell cycle genes and Notch signaling provides further insight into the complex interplay between cell proliferation and differentiation in the fly (Kugler, 2007).

Requirements for mediator complex subunits distinguish three classes of notch target genes at the Drosophila wing margin

Spatial and temporal gene regulation relies on a combinatorial code of sequence-specific transcription factors that must be integrated by the general transcriptional machinery. A key link between the two is the mediator complex, which consists of a core complex that reversibly associates with the accessory kinase module. Genes activated by Notch signaling at the dorsal-ventral boundary of the Drosophila wing disc fall into three classes that are affected differently by the loss of kinase module subunits. One class requires all four kinase module subunits for activation, while the others require only Med12 and Med13, either for activation or for repression. These distinctions do not result from different requirements for the Notch coactivator Mastermind or the corepressors Hairless and Groucho. It is proposed that interactions with the kinase module through distinct cofactors allow the DNA-binding protein Suppressor of Hairless to carry out both its activator and repressor functions (Janody, 2011).

Intercellular signaling pathways drive many processes during development. Their activation results in changes in transcription factor activity that lead to the activation or repression of specific target genes. An important goal is to understand the transcriptional regulatory codes that allow the combinations of proteins bound to enhancer elements to direct precise patterns of gene expression. One well-characterized developmental paradigm is the specification of the Drosophila wing margin by Notch signaling. The Notch receptor is specifically activated at the dorsal-ventral boundary of the larval wing imaginal disc, due to the restricted expression of its ligands Delta and Serrate and of the glycosyltransferase Fringe. Notch activation results in expression of the target genes Enhancer of split m8 (E(spl)m8), cut, wingless (wg), and vestigial (vg), the last through a specific enhancer element known as the boundary enhancer (vgBE). Wg signaling then leads to the differentiation of characteristic sensory bristles adjacent to the margin of the adult wing (Janody, 2011).

Upon ligand binding, Notch is cleaved by the γ-secretase complex, and its intracellular domain (Nintra) enters the nucleus, where it interacts with the DNA-binding protein Suppressor of Hairless (Su(H)). In the absence of Notch activation, Su(H) represses target gene expression through interactions with the corepressor Hairless (H), which binds to Groucho (Gro) and C-terminal binding protein (CtBP). Nintra displaces these corepressors from Su(H) and recruits coactivators such as Mastermind (Mam). It has been proposed that only a subset of Notch target genes require Su(H) to recruit coactivators, while others require Notch signaling only to relieve Su(H)-mediated repression, allowing transcription to be activated by other factors. However, the mechanisms by which Su(H) directs both activation and repression are not fully understood (Janody, 2011).

The mediator complex is thought to promote transcriptional activation by recruiting RNA polymerase II (Pol II), the general transcriptional machinery, and the histone acetyltransferase p300 to promoters, and by stimulating transcriptional elongation by Pol II molecules paused downstream of the promoter. The 'head' and 'middle' modules of the core complex bind to Pol II and general transcription factors, while the 'tail' module consists largely of adaptor subunits that bind to sequence-specific transcription factors. This core complex reversibly associates with a fourth 'kinase' module that consists of the four subunits Med12, Med13, Cdk8, and Cyclin C (CycC). Several studies have implicated the kinase module in transcriptional repression, which can be mediated by phosphorylation of Pol II and other factors by Cdk8, by histone methyltransferase recruitment, and by occlusion of the Pol II binding site. However, this module also appears to function in activation in some contexts; for example, it promotes Wnt target gene expression during Drosophila and mouse development, in mammalian cells, and in colon cancer. Although all four subunits have very similar mutant phenotypes in yeast, loss of Med12 or Med13 has more severe effects on Drosophila development than loss of Cdk8 or CycC, suggesting that Med12 and Med13 have evolved additional functions in higher eukaryotes (Janody, 2011).

This study shows that Notch target genes at the wing margin can be divided into three classes based on their requirements for kinase module subunits. An E(spl)m8 reporter requires all four subunits for its activation, cut requires only Med12 and Med13 (known as Kohtalo [Kto] and Skuld [Skd], respectively, in Drosophila) for its activation, and wg and the vgBE enhancer require Med12 and Med13 for their repression in cells close to the wing margin. Because Med12 and Med13 coimmunoprecipitate with Su(H), regulate an artificial reporter driven by Su(H) binding sites, and can be replaced by a VP16 activation domain or a WRPW repression signal fused to Su(H), it is proposed that the kinase module directly regulates Notch target genes. All four Notch target genes fail to be expressed in the absence of Mam and are similarly affected by the loss of Hairless or Gro, suggesting that other more specific cofactors might recruit kinase module subunits to these genes (Janody, 2011).

The kinase module of the mediator complex is conserved throughout eukaryotes, yet its functions in transcription remain poorly understood. In yeast, loss of any of the four subunits has a very similar effect. In Drosophila, however, loss of Med12 or Med13 has more dramatic effects than loss of Cdk8 or CycC. The kinase module was originally thought to be primarily important for transcriptional repression, mediated by the kinase activity of Cdk8. However, Med12 and Med13 appear to directly activate genes regulated by Wnt signaling in Drosophila and mammalian systems, and also play a positive role in gene activation by the Gli3 and Nanog transcription factors. The data presented in this study confirm that Med12 and Med13 have functions distinct from Cdk8 and CycC. In addition, evidence is provided that all four kinase module subunits contribute to the activation of E(spl)m8 (Janody, 2011).

The human Mastermind homologue MAM has been shown to recruit Cdk8 and CycC to promoters of Notch target genes, where Cdk8 phosphorylates the intracellular domain of Notch, leading to its ubiquitination by the Fbw7 ligase and degradation (Fryer, 2004). This mechanism would be expected to reduce Notch target gene expression, consistent with the increase in E(spl)mβ expression seen in clones lacking the Drosophila Fbw7 homologue Archipelago (Nicholson, 2011); thus it cannot explain the positive effects of Cdk8 and CycC on E(spl)m8. A function for Cdk8 and CycC in Notch-mediated activation would be analogous to recent findings showing that Cdk8 phosphorylation of Smad transcription factors and of histone H3 promotes activation. Cdk8 phosphorylation of RNA polymerase II (Pol II) is also important for transcriptional elongation (Janody, 2011).

Of interest, the current data also suggest that Med12 and Med13 are involved in the repression of wg and the vgBE enhancer in the absence of Notch signaling. The kinase module has been proposed to inhibit transcription through steric hindrance of Pol II binding, independently of Cdk8 kinase activity. Removal of this module on the C/EBP promoter is thought to convert the mediator complex to its active form. In contrast, this study find that wg and vgBE require Med12 and Med13 for their repression but not their activation, while cut and E(spl)m8 require Med12 and Med13 only for their activation, arguing that the two functions occur on different promoters. It cannot be ruled out that Med12 and Med13 have only indirect effects on some of the genes examined; however, their physical association with Su(H) and the requirement for Su(H) binding sites for misexpression of an artificial reporter in skd and kto mutant clones are consistent with a direct effect of Med12 and Med13 on the Su(H) complex (Janody, 2011).

Med12 and Med13 are found associated with both active and inactive promoters in genome-wide chromatin immunoprecipitation studies, suggesting that they can have different effects on transcription when bound to distinct interaction partners. Although both are very large proteins, they contain no domains predicted to have enzymatic activity, and may instead act as scaffolds for the assembly of transcriptional complexes (Janody, 2011).

It has been proposed that Notch target genes could be categorized into two classes: permissive genes, for which the primary function of Notch is to relieve repression by the Su(H) complex, and instructive genes, for which Notch plays an essential role in activation by recruiting specific coactivators. These differences presumably depend on the combinatorial code of transcription factors that regulate each promoter. This study shows that vgBE, an enhancer previously placed in the permissive category, as well as wg, require Med12 and Med13 for their repression but not their activation. During eye development, the proneural gene atonal is likewise regulated permissively by Notch, and ectopically expressed in skd or kto mutant clones. Unexpectedly, this study found that Gro, previously thought to be a cofactor through which Hairless mediates repression, is not required for the repression of vgBE or wg. Hairless may repress target genes at the wing margin through CtBP, its other binding partner. Alternatively, Gro may affect the expression of other upstream regulators of wing margin fate, masking its repressive effect on the genes that were examined (Janody, 2011).

It was also show in this study that instructive Notch target genes can be further subdivided into two classes based on their requirement for kinase module subunits; E(spl)m8 requires all four subunits, while cut requires Med12 and Med13, but not Cdk8 and CycC. Cdk8 and CycC may simply increase the ability of the mediator complex to recruit Pol II or promote transcriptional initiation; this model would suggest that E(spl)m8 has a higher activation threshold than cut. Alternatively, Cdk8 and CycC might enhance the function of a transcription factor that is specifically required for the expression of E(spl)m8 but not cut. Good candidates for such factors would be the proneural proteins Achaete or Scute or their partner Daughterless (Janody, 2011).

The mechanism by which the kinase module is recruited to promote the activation of instructive target genes is not yet clear. Although Mam proteins are well-characterized coactivators for N_intra, this study found that Mam is necessary for the activation of both instructive and permissive genes. It may thus have a general function in transcriptional activation, such as recruiting histone acetyltransferases or stabilizing the Notch-Su(H) complex. A coactivator that recruits Med12 and Med13 specifically to instructive target genes to promote activation may remain to be identified. The current results, like recent reports demonstrating that the arrangement of Su(H) binding sites can affect the interactions between Notch and its coactivators, highlight the complexity in the mechanisms through which promoter elements respond to Notch signaling (Janody, 2011).

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